Amino acid ester and amide renin inhibitor

ABSTRACT

Compounds of the formula  &lt;IMAGE&gt;  wherein A is  &lt;IMAGE&gt;  intervene in the conversion of angiotensinogen to angiotensin II by inhibiting renin and thus are useful as anti-hypertensive agents.

PRIOR APPLICATION

This application is a continuation-in-part of U.S. Ser. No. 666,015filed Oct. 29, 1984, now abandoned.

BACKGROUND OF THE INVENTION

Szelke et al. in European Patent Application No. 104,041 disclose renininhibitory polypeptides including the partial sequence X--A--B--Z--W

wherein

A is ##STR3## and G is ##STR4## X is hydrogen, protecting group or anamino acyl residue, B is a lipophilic amino acyl residue, and

Z plus W are an amino alcohol residue or Z is aminoacyl and W ishydroxy, ester, amide, etc.

Natarajan et al. in U.S. Pat. No. 4,470,973 disclose aminoketonecarboxylic acids of the formula ##STR5## as intermediates in thepreparation of aminoketone peptides which possess angiotensin convertingenzyme or enkephalinase inhibition activity.

Gordon et al. in U.S. application Ser. No. 515,729 filed July 21, 1983now U.S. Pat. No. 4,514,391 disclose hydroxy substituted peptidecompounds of the formula ##STR6## which possess angiotensin convertingenzyme or enkephalinase inhibition activity.

SUMMARY OF THE INVENTION

This invention is directed to new amino acid ester and amide compoundsof formula I including pharamaceutically acceptable salts thereof##STR7## A is ##STR8## R₄ is --O--lower alkyl, --O--(CH₂)_(m) --aryl,--O--(CH₂)_(m) --heterocyclo, or ##STR9## R₅ is hydrogen, lower alkyl,--(CH₂)_(m) --aryl, or --(CH₂)_(m) --heterocyclo.

m is zero, one, two, three or four.

R₆ is lower alkyl, --(CH₂)_(m) --aryl, --(CH₂)_(m) --heterocyclo, or##STR10## R₈ is hydrogen, ##STR11## R₁₀ is hydrogen, ##STR12## R₂, R₃,R₇ and R₉ are independently selected from the group consisting ofhydrogen, lower alkyl, halo substituted lower alkyl, --(CH₂)_(n) --aryl,--(CH₂)_(n) --heterocyclo, --(CH₂)_(n) --OH, --(CH₂)_(n) --NH₂,--(CH₂)_(n) --SH, --(CH₂)_(n) --S--lower alkyl, --(CH₂)₂ --S--(CH₂)₂--NH₂, ##STR13## n is an integer from 1 to 4. R₁₁ is ##STR14## R₁ ishydrogen, lower alkyl, --(CH₂)_(n) --aryl, --(CH₂)_(n) --cycloalkyl,##STR15##

DETAILED DESCRIPTION OF THE INVENTION

This invention in its broadcast aspects relates to the amino acid esterand amide compounds of formula I above, to compositions and the methodof using such compounds as antihypertensive agents.

The term lower alkyl used in defining various symbols refers to straightor branched chain radicals having up to seven carbons, preferably fromone to four carbons. Similarly, the terms lower alkoxy and loweralkylthio refer to such lower alkyl groups attached to an oxygen orsulfur.

The term cycloalkyl refers to saturated rings of 4 to 7 carbon atomswith cyclopentyl and cyclohexyl being most preferred.

The term halogen refers to chloro, bromo and fluoro.

The term halo substituted lower alkyl refers to such lower alkyl groupsdescribed above in which one or more hydrogens have been replaced bychloro, bromo or fluoro groups such as trifluoromethyl, which ispreferred, pentafluoroethyl, 2,2,2-trichloroethyl, chloromethyl,bromoethyl, etc.

The term aryl refers to phenyl, 1-naphthyl, 2-naphthyl, mono substitutedphenyl, 1-naphthyl, or 2-naphthyl wherein said substituent is loweralkyl of 1 to 4 carbons, lower alkylthio of 1 to 4 carbons, lower alkoxyof 1 to 4 carbons, halogen, hydroxy, amino, --NH-alkyl wherein alkyl isof 1 to 4 carbons, or --N(alkyl)₂ wherein alkyl is of 1 to 4 carbons, dior tri substituted phenyl, 1-naphthyl or 2-naphthyl wherein saidsubstituents are methyl, methoxy, methylthio, halogen, or hydroxy.

The term heterocyclo refers to fully saturated or unsaturated rings of 5or 6 atoms containing one or two O and S atoms and/or one to four Natoms provided that the total number of hetero atoms in the ring is 4 orless. The heterocyclo ring is attached by way of an available carbonatom. Preferred heterocyclo groups include 2- and 3-thienyl, 2- and3-furyl, 2-, 3- and 4-pyridyl, and imidazolyl. The term heterocyclo alsoincludes bicyclic rings wherein the five or six membered ring containingO, S and N atoms as defined above is fused to a benzene ring. Thepreferred bicyclic ring is indolyl.

Compounds of formula I wherein A is ##STR16## can be prepared asfollows. A halomethyl ketone of the formula ##STR17## wherein R₄₀ is aprotecting group such as ##STR18## especially wherein halo is Cl, isreacted with an amine of the formula ##STR19## This reaction isperformed in the presence of sodium bicarbonate and dimethylformamideand gives the compound of the formula ##STR20##

The intermediate of formula IV is then treated to remove the R₄₀protecting group such as by treatment with hydrochloric acid in thepresence of ethyl acetate and the resulting amine hydrochloride salt isreacted with the carboxylic acid of the formula

    R.sub.6 --COOH                                             (V)

in the presence of dicyclohexylcarbodiimide and hydroxybenzotriazole togive the desired final product.

The compounds of formula I wherein A is ##STR21## can be prepared asfollows. The intermediates of formula IV is treated with a conventionalreducing agent such as sodium borohydride, sodium cyanoborohydride,diisobutyl aluminum hydride, lithium tri t-butoxy aluminum hydride,etc., to give ##STR22##

The intermediate of formula VI is then treated to remove the R₄₀protecting group and the resulting amine is reacted with the carboxylicacid of formula V as described above to give the desired final products.

The compounds of formula I wherein A is ##STR23## can also be preparedby reacting an oxazolidine, trimethylsilylethyl ester of the formula##STR24## so as to introduce the desired R₄ substituent. For example,when R₄ is NH₂ the ester of formula VII is treated with tetra n-butylammonium fluoride, followed by isobutylchloroformate, and finallymethanolic ammonia, and when R₄ is --NH--R₅ the ester of formula VII istreated with tetra n-butyl ammonium fluoride, followed byisobutylchloroformate, and finally the amine H₂ N-R₅. The resultingoxazolidine is then treated with trifluoroacetic acid and aqueoushydrochloric acid to give the intermediate of the formula ##STR25##

The intermediate of formula VIII is then treated with the carboxylicacid of formula V in the presence of dicyclohexylcarbodiimide andhydroxybenzotriazole as described above to give the desired finalproducts.

In the above reactions, if R₁ is hydrogen then that N-atom would beprotected for example by reacting the intermediate of formula IV withbenzyloxycarbonyl chloride in the presence of pyridine and benzene. Thebenzyloxycarbonyl group could then be removed as the last step of thesynthesis by hydrogenation in the presence of palladium on carboncatalyst.

Similarly, if any of R₁, R₂, R₃, R₇ and R₉ in the above reactions are--(CH₂)_(n) --aryl wherein aryl is phenyl, 1-naphthyl, 2-naphthylsubstituted with one or more hydroxy or amino groups, --(CH₂)_(n)--heterocyclo wherein heterocyclo is an imidazolyl, --(CH₂)_(n) --NH₂,--(CH₂)_(n) --SH, --(CH₂)_(n) --OH, or ##STR26## then the hydroxyl,amino, imidazolyl, mercaptan, or guanidinyl function should be protectedduring the reaction. Suitable protecting groups includebenzyloxycarbonyl, t-butoxycarbonyl, benzyl, benzhydryl, trityl, etc.,and nitro in the case of guanidinyl. The protecting group is removed byhydrogenation, treatment with acid, or by other known means followingcompletion of the reaction.

The starting materials of formula II can be prepared by reacting anN-protected amino acid of the formula ##STR27## withisobutylchloroformate, followed by diazomethane, and finallyhydrochloric acid.

The oxazolidine, trimethylsilyl ethyl ester of formula VII can beprepared by reacting the halomethyl ketone of formula II with the aminoacid, trimethylsilyl ester of formula ##STR28## in the presence ofsodium iodide, sodium bicarbonate and dimethylformamide to give thecompound of the formula ##STR29##

The ketone of formula XI is reduced to the corresponding alcohol bytreatment with sodium borohydride or other reducing agents as describedabove. This alcohol is then treated with 2-methoxy-propene in thepresence of a catalytic amount of pyridinium-p-toluenesulfonic acid togive the desired oxazolidine, trimethylsilyl ethyl ester.

The various peptide intermediates employed in above procedures are knownin the literature or can be readily prepared by known methods. See forexample, The Peptides, Volume 1, "Major Methods Of Peptide BondFormation", Academic Press (1979).

Preferred compounds of this invention are those of formula I wherein:

A is ##STR30## R₁ is hydrogen, ##STR31## especially hydrogen. R₂ isstraight or branched chain lower alkyl of 1 to 4 carbons, benzyl,phenethyl, or ##STR32## especially --CH₂ --CH(CH₃)₂. R₃ is hydrogen,straight or branched chain lower alkyl of 1 to 4 carbons, benzyl orphenethyl, especially --CH₂ --CH(CH₃)₂ or --CH(CH₃)₂.

R₄ is --NH₂, --NH--(CH₂)_(n) --aryl, or --NH--(CH₂)_(n) --heterocyclowherein n is one, two, or three, aryl is phenyl, and heterocyclo ispyridyl. ##STR33## R₇ is ##STR34## R₈ is ##STR35## R₉ is --(CH₂)_(n)-aryl wherein aryl is phenyl or 1-naphthyl and n is one or two,especially ##STR36## R₁₀ is ##STR37##

The compounds of formula I form salts with a variety of inorganic andorganic acids. The non-toxic pharmaceutically acceptable salts arepreferred, although other salts are also useful in isolating orpurifying the product. Such pharmaceutically acceptable salts includethose formed with hydrochloric acid, methanesulfonic acid, sulfuricacid, acetic acid, maleic acid, etc. The salts are obtained by reactingthe product with an equivalent amount of the acid in a medium in whichthe salt precipitates.

The compounds of formula I contain asymmetric centers when any or all ofR₂, Rhd 3, R₇, and R₉ are other than hydrogen. An additional asymmetriccenter is present when A is ##STR38## Thus, the compounds of formula Ican exist in diasteroisomeric forms or in mixtures thereof. The abovedescribed processes can utilize racemates, enantiomers or diastereomersas starting materials. When diastereomeric products are prepared, theycan be separated by conventional chromatographic or fractionalcrystallization methods.

The compounds of formula I, and the pharmaceutically acceptable saltsthereof, are antihypertensive agents. They inhibit the conversion ofangiotensinogen to angiotensin I and therefore, are useful in reducingor relieving angiotensin related hypertension. The action of the enzymerenin on angiotensinogen, a pseudoglobulin in blood plasma, producesangiotensin I. Angiotensin I is converted by angiotensin convertingenzyme (ACE) to angiotensin II. The latter is an active pressorsubstance which has been implicated as the causative agent in severalforms of hypertension in various mammalian species, e.g., humans. Thecompounds of this invention intervene in theangiotensinogen→(renin)→angiotensin I→(ACE)→angiotensin II sequence byinhibiting renin and reducing or eliminating the formation of thepressor substance angiotensin II. Thus by the administration of acomposition containing one (or a combination) of the compounds of thisinvention, angiotensin dependent hypertension in a species of mammal(e.g., humans) suffering therefrom is alleviated. A single dose, orpreferably two to four divided daily doses, provided on a basis of about100 to 1000 mg., preferably about 250 to 500 mg. per kg. of body weightper day is appropriate to reduce blood pressure. The substance ispreferably administered orally, but parenteral routes such as thesubcutaneous, intramuscular, intraveneous or intraperitoneal routes canalso be employed.

The compounds of this invention can also be formulated in combinationwith a diuretic for the treatment of hypertension.

A combination product comprising a compound of this invention and adiuretic can be administered in an effective amount which comprises atotal daily dosage of about 1000 to 6000 mg., preferably about 3000 to4000 mg. of a compound of this invention, and about 15 to 300 mg.,preferably about 15 to 200 mg. of the diuretic, to a mammalian speciesin need thereof. Exemplary of the diuretics contemplated for use incombination with a compound of this invention are the thiazidediuretics, e.g., chlorothiazide, hydrochlorothiazide, flumethiazide,hydroflumethiazide, bendroflumethiazide, methyclothiazide,trichloromethiazide, polythiazide or benzthiazide as well as ethacrynicacid, ticrynafen, chlorthalidone, furosemide, musolimine, bumetanide,triamterene, amiloride and spironolactone and salts of such compounds.

The compounds of formula I can be formulated for use in the reduction ofblood pressure in compositions such as tablets, capsules or elixirs fororal administration or in sterile solutions or suspensions forparenteral administration. About 100 to 500 mg. of a compound of formulaI is compounded with physiologically acceptable vehicle, carrier,excipient, binder, preservative, stabilizer, flavor, etc., in a unitdosage form as called for by accepted pharmaceutical practice. Theamount of active substance in these compositions or preparations is suchthat a suitable dosage in the range indicated is obtained.

The following examples are illustrative of the invention. Temperaturesare given in degrees centrigrade.

EXAMPLE 1 N²-[3-[[N-[N-[(1,1-Dimethylethoxy)carbonyl]-L-phenylalanyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-N-[2-(2-pyridinyl)ethyl]-L-leucinamide,acetic acid salt (1:2) (a) 3-[(Phenylmethoxy)methyl]-L-histidine, methylester, dihydrochloride

N-[(1,1-Dimethylethoxy)carbonyl]-3-[(phenylmethoxy)methyl]-L-histidine,methyl ester, monohydrochloride (7.8 g., 18.3 mmole) [prepared accordingto the procedure of Brown et al., J. Chem. Soc. Perins Trans., Vol. 1,p. 2261 (1979)] is suspended in ethyl acetate (140 ml.) under a flow ofnitrogen, cooled in an ice-water bath. Dry hydrogen chloride is bubbledin to saturation and the resulting solution is stoppered and kept coldfor 20 minutes and then at ambient temperature for 40 minutes. Thereaction mixture is then concentrated in vacuo to give 8.4 g. of crudeproduct. Recrystallization from hot isopropanol yields 5.2 g. of3-[(phenylmethoxy)methyl]-L-histidine, methyl ester, dihydrochloride;m.p. 159°-160°, resolidified at m.p. 209°-210°. [α]_(D) =+13.4° (c=1.11,methanol).

(b)N-[N-[(1,1-Dimethylethoxy)carbonyl]-L-phenylalanyl]-3-[(phenylmethoxy)methyl]-L-histidine,methyl ester

Dicyclohexylcarbodiimide (2.17 g., 10.5 mmole) is added to a mixture of3-[(phenylmethoxy)methyl]-L-histidine, methyl ester, dihydrochloride(3.62 g., 10 mmole), N-[(1,1-dimethylethoxy)carbony]-L-phenylalanine(2.56 g., 10 mmole), and hydroxybenzotriazole hydrate (1.68 g., 11mmole) in dimethylformamide (70 ml.) cooled in an ice-water bath undernitrogen followed by the addition of N-methylmorpholine (2.02 g., 20mmole). The reaction is allowed to warm to room temperature overnight,then chilled for 15 minutes in an ice-bath after diluting with 200 ml.of ethyl acetate. The cold solution is filtered and the filtrate iswashed with three 70 ml. portions of water, saturated sodium bicarbonateand brine, dried (MgSO₄), and concentrated in vacuo to give 4.9 g. ofcrude product. Flash chromatography on 250 g. of LPS-1 silica geleluting with 3 column volumes of 1:1 chloroform:ethyl acetate followedby a gradient to 15:1 chloroform:methyl yields 4.2 g. of purifiedproduct. Recrystallization from hot ethyl acetate affords 3.7 g. ofN-[N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalanyl]-3-[(phenylmethoxy)methyl]-L-histidine,methyl ester; m.p. 165°-166°; [α]_(D) =-15.4° (c=0.5, methanol). TLC(silica gel; chloroform:methanol 12:1) R_(f) =0.40.

(c)N-[N-[(1,1-Dimethylethoxy)carbonyl]-L-phenylalanyl]-3-[(phenylmethoxy)methyl]-L-histidine

1N Aqueous sodium hydroxide (6.8 ml.) is added to a solution ofN-[N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalanyl]-3-[(phenylmethoxy)methyl]-L-histidine,methyl ester (3.3 g., 6.1 mmole) in methanol (18 ml.). After 3 hours,the reaction is diluted with water (42 ml.) and then concentrated invacuo to remove most of the methanol. The resulting solution is rinsedwith 25 ml. of ether and then acidified to pH of 4.5 using 1Nhydrochloric acid. The precipitated solid is filtered, washed withwater, and dried in vacuo to give 2.95 g. ofN-[N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalanyl]-3-[(phenylmethoxy)methyl]-L-histidine;m.p. 189°-190° (dec.); [α]_(D) =-5.7° (c=1, dimethylformamide). TLC(silica gel; 2% NH₄ OH in n-propanol) R_(f) =0.36.

(d) [(Phenylmethoxy)carbonyl]-L-leucine, 2-(trimethylsilyl)ethyl ester

A mixture of [(phenylmethoxy)carbonyl]-L-leucine (20.1 g., 75.7 mmole),dimethylaminopyridine (0.925 g., 7.57 mmole), and2-(trimethylsilyl)ethanol (8.95 g., 75.7 mmole) in methylene chloride(200 ml.) is cooled in an ice-bath under nitrogen and treated with asolution of dicyclohexylcarbodiimide (15.6 g., 75.7 mmole) in 50 ml. ofmethylene chloride. The ice-bath is removed after 20 minutes and thereaction is allowed to come to room temperature overnight. The reactionmixture is filtered, concentrated in vacuo and partitioned between 800ml. of ether and 200 ml. of water. The organic layer is separated andfurther rinsed with saturated sodium bicarbonate, water, 10% potassiumbisulfate, water, and brine, dried (MgSO₄), and concentrated in vacuo togive 26.1 g. of crude product. Flash filtration over silica gel (180 g.in 20:1 hexane:ethyl acetate) yields 22.9 g. of crude product which ischromatographed using the Waters Prep 500 LC, two columns eluted with15:1 hexane-ethyl acetate (250 ml./minute, 200 ml. fractions). Combiningthe pure product fractions yields 19.6 g. of[(phenylmethoxy)carbonyl]-L-leucine, 2-(trimethylsilyl)ethyl ester as anoil; [α]_(D) =-7.3° (c=1, chloroform). TLC (silica gel; 10:1hexane:ethyl acetate) R_(f) =0.12.

(e) L-Leucine, 2-(trimethylsilyl)ethyl ester

1 g. of 10% palladium on carbon catalyst is added to a solution of[(phenylmethoxy)carbonyl]-L-leucine, 2-(trimethylsilyl)ethyl ester (9.1g., 24.9 mmole) in ethyl acetate (200 ml.) cooled in an ice-bath undernitrogen. The reaction mixture is subjected to a steady stream ofhydrogen gas for 2.5 hours at room temperature, then filtered andconcentrated in vacuo to remove volatiles and yields 5.8 g. ofL-leucine, 2-(trimethylsilyl)ethyl ester as a liquid product. TLC(silica gel, 3:1 hexane:ethyl acetate) R_(f) =0.08.

(f) [(S)-1-Chloro-5-methyl-2-oxo-3-hexyl]carbamic acid,1,1-dimethylethyl ester

N-Methyl-N'-nitro-N-nitrosoguanidine (24.2 g., 165 mmole) is addedportionwise over a period of 30 minutes to a mixture of ether (275 ml.)and 40% aqueous potassium hydroxide (75 ml.) in a 1 l. Erlenmeyer flaskcooled in a ice-water bath. Throughout the reaction the flask ismagnetically stirred and loosely stoppered. At the end of the addition,the reaction is stirred cold for 45 minutes longer.

A solution of [(1,1-dimethylethoxy)carbonyl]-L-leucine hydrate (19.2 g.,77 mmole) in dry tetrahydrofuran (180 ml.) is cooled to -10° to -15°under an atmosphere of argon. The reaction mixture is treated withN-methylmorpholine (7.79 g., 77 mmole), added neat, followed by thecareful addition of isobutylchloroformate (10.5 g., 77 mmole), addedneat and dropwise while keeping the temperature between -10° and -15°.Upon completion of the addition, the solution is kept at -15°.

As much as possible of the diazomethane solution in ether is decantedinto a 1 l. Erlenmeyer flask and kept cold. The remainder is poured intoa 500 ml. separatory funnel. The aqueous layer is drained and discardedwhile the ether is added to the decanted material. The total etherealdiazomethane is quickly dried over solid potassium hydroxide, thendecanted into a 1 l. filtering flask fitted with an argon sidearmconnection, magnetically stirred and cooled in a -10° bath. Total volumeby now is about 400 ml. A filtering funnel is put in place with vacuummomentarily connected at sidearm, the mixed anhydride preparation isquickly filtered directly into the -10° ethereal diazomethane. Etherrinses are used to finish the transfer. The vacuum is replaced by theargon line and the reaction is allowed to warm to 0° and is kept at 0°for one hour. The flask is then stoppered, fitted with a balloon andrefrigerated overnight.

The reaction mixture is next rinsed with 400 ml. each of 3% aqueousacetic acid, water, saturated sodium bicarbonate, water, and brine,dried (MgSO₄) and concentrated in vacuo. The crude product isrecrystallized from etherpetroleum ether to give 17.2 g. of[(S)-1-chloro-5-methyl-2-oxo-3-hexyl]carbamic acid, 1,1-dimethylethylester; m.p. 87°-89°; [α]_(D) =-51.2° (c=1, methylene chloride).

Calculated for C₁₂ H₂₁ N₃ O₃ : C, 56.45; H, 8.29; N, 16.46 Found: C,56.13; H, 8.31; N, 16.39.

(g)N-[(S)-3-[[(1,1-Dimethylethoxy)carbonyl]amino]-5-methyl-2-oxohexyl]-L-leucine,2-(trimethylsilyl)ethyl ester

A solution of [(S)-1-chloro-5-methyl-2-oxo-3-hexyl]carbamic acid,1,1-dimethylethyl ester (3.55 g., 13.5 mmole) in dimethylformamide (18ml.) is added, in one portion, to a mixture of L-leucine,2-(trimethylsilyl)ethyl ester (5.47 g., 23.6 mmole), sodium iodide (1.06g., 7.1 mmole), and sodium bicarbonate (2.0 g., 23.6 mmole) indimethylformamide (18 ml.). The reaction, under an atmosphere ofnitrogen, is stirred at ambient temperature overnight, then diluted with500 ml. of 1:1 ethyl acetate:ether and washed with 100 ml. portions ofwater, 5% sodium bicarbonate, water, and brine, dried (MgSO₄) andconcentrated in vacuo to give 7.8 g. of crude product. Flashchromatography on 85 g. of LPS-1 silica gel eluting with 10:1hexane:ethyl acetate yields 5.0 g. ofN-[(S)-3-[[(1,1-dimethylethoxy)carbonyl]amino]-5-methyl-2-oxohexyl]-L-leucine,2-(trimethylsilyl)ethyl ester as an oil; [α]_(D) =-15.5° (c=1,chloroform). TLC (silica gel; 6:1 hexane:ethyl acetate) R_(f) =0.11.

(h)N-[(S)-3-[[(1,1-Dimethylethoxy)carbonyl]amino]-5-methyl-2-hydroxyhexyl]-L-leucine,2-(trimethylsilyl)ethyl ester

Sodium borohydride (2.1 g., 54.5 mmole) is added to a solution ofN-[(S)-3-[[(1,1-dimethylethoxy)carbonyl]amino]-5-methyl-2-oxohexyl]-L-leucine,2-(trimethylsilyl)ethyl ester (5.0 g., 10.9 mmole) in a mixture oftetrahydrofuran (150 ml.) and water (50 ml.) cooled in an ice-waterbath. After 5 minutes the reaction is poured into water (300 ml.) andextracted with ethyl acetate (600 ml.). The organic extract is rinsedfurther with water and brine, dried (MgSO₄) and concentrated in vacuo toyield 4.7 g. of crude product. Flash chromatography on 140 g. of LPS-1silica gel eluting with 10:1 petroleum ether:acetone yields 3.5 g. ofN-[(S)-3-[[(1,1-dimethylethoxy)carbonyl]amino]-5-methyl-2-hydroxyhexyl]-L-leucine,2-(trimethylsilyl)ethyl ester as an oil; [α]_(D) =-27.8°; (c=1,chloroform). TLC (silica gel; 10:1 petroleum ether:acetone) R_(f) =0.20.

(i)N-[(S)-3-[[(1,1-Dimethylethoxy)carbonyl]amino]-5-methyl-2-hydroxyhexyl]-N-[(phenylmethoxy)carbonyl]-L-leucine,2-(trimethylsilyl)ethyl ester

A mixture ofN[(S)-3-[[(1,1-dimethylethoxy)carbonyl]amino]-5-methyl-2-hydroxyhexyl]-L-leucine,2-(trimethylsilyl)ethyl ester (3.5 g., 7.6 mmole) andN-[(phenylmethoxy)carbonyloxy]succinimide (2.8 g., 11.4 mmole) in drytetrahydrofuran (13.5 ml.) is stirred under nitrogen in a stopperedflask at ambient temperature for 48 hours, then diluted with 100 ml. ofether and rinsed with water and brined, dried (MgSO₄) and concentratedin vacuo to give 5.6 g. of crude product. The mixture is flash filteredthrough 60 g. of LPS-1 silica gel eluting with 20:1 petroleumether:acetone to give 4.3 g. ofN-[(S)-3-[[(1,1-dimethylethoxy)carbonyl]amino]-5-methyl-2-hydroxyhexyl]-N-[(phenylmethoxy)carbonyl]-L-leucine,2-(trimethylsilyl)ethyl ester as a mixture of isomers.

(j)N-[[(3S)-3-[(1,1-Dimethylethoxy)carbonyl]-2,2-dimethyl-4-(2-methylpropyl)oxazolidin-5-yl]methyl]-N-[(phenylmethoxy)carbonyl]-L-leucine,2-(trimethylsilyl)ethyl ester

N-[(S)-3-[[(1,1-Dimethylethoxy)carbonyl]amino]-5-methyl-2-hydroxyhexyl]-N-[(phenylmethyoxy)carbonyl]-L-leucine,2-(trimethylsilyl)ethyl ester (1.9 g., 3.2 mmole) is dissolved in drymethylene chloride (7 ml.) and treated with 12-methoxypropene (0.690 g.,9.6 mmole) followed by pyridinium-p-toluenesulfonic acid. The reactionis stirred at room temperature under nitrogen for five hours and thendiluted with 500 ml. of ether and rinsed with two 75 ml. portions ofwater and 75 ml. of brine, dried (MgSO₄) and concentrated in vacuo togive the crude product. Flash chromatography on 100 g. of LPS-1 silicagel eluting with a gradient from 100:1 to 25:1 petroleum ether:acetonegives 1.9 g. ofN-[[(3S)-3-[(1,1-dimethylethoxy)carbonyl]-2,2-dimethyl-4-(2-methylpropyl)oxazolidin-5-yl]methyl]-N-[(phenylmethoxy)carbonyl]-L-leucine,2-(trimethylsilyl)ethyl ester.

(k) N²-[[(3S)-3-[(1,1-Dimethylethoxy)carbonyl]-2,2-dimethyl-4-(2-methylpropyl)oxozolidin-5-yl]methyl]-N-[(phenylmethoxy)carbonyl]-N-[2-(2-pyridinyl)ethyl]-L-leucinamide

A solution of the 2-(trimethylsilyl) ethyl ester product from part (j)(940 mg., 1.48 mmole) in dimethylformamide (11 ml.) at room temperatureunder argon is treated with tetra n-butyl ammonium fluoride trihydrate(934 mg., 2.96 mmole). After 15 minutes, the reaction is worked up bydiluting with ether (50 ml.) and ethyl acetate (50 ml.), rinsed withthree 25 ml. portions of water, rinsed with brine, dried (MgSO₄), andconcentrated in vacuo to give 765 mg. of the free acid intermediate.

A solution of this free acid (390 mg., 0.729 mmole),hydroxybenzotriazole hydrate 112 mg., 0.729 mmole) and2-(2-aminoethyl)pyridine (98 mg., 0.802 mmole) in dimethylformamide (7ml.) is cooled in an ice-water bath under argon and treated withdicyclohexylcarbodiimide (158 mg., 0.765 mmole). The reaction is allowedto come to room temperature overnight, then diluted with 30 ml. of ethylacetate and chilled in an ice-water bath for 15 minutes. The chilledmixture is then filtered and diluted with 30 ml. of ether. The organicsolution is rinsed with three 10 ml. portions of water and 10 ml. ofbrine, dried (MgSO₄) and concentrated in vacuo to give 539 mg. of crudeproduct. Flash chromatography on 42 g. LPS-1 silica gel packed inchloroform and eluted with 20:1 chloroform:methanol yields partiallypurified product. Rechromatography on 40 g. of LPS-1 silica gel packedin 1:1 chloroform:hexanes and eluted with a gradient of 100:1 to 20:1chloroform:methanol yields 422 mg. of N²-[[(3S)-3-[(1,1-dimethylethoxy)carbonyl]-2,2-dimethyl-4-(2-methylpropyl)oxazolidin-5-yl]methyl]-N-[(phenylmethoxy)carbonyl]-N-[2-(2-pyridinyl)ethyl]-L-leucinamide.TLC (silica gel, 10:1 chloroform:methanol) R_(f) =0.47.

(l) N²-[3-[[N-[N-[(1,1-Dimethylethoxy)carbonyl]-L-phenylalanyl]-3-[(phenylmethoxy)methyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-N-[(phenylmethoxy)carbonyl]-N-[2-(2-pyridinyl)ethyl]-L-leucinamide

A solution of N²-[[(3S)-3-[(1,1-dimethylethoxy)carbonyl]-2,2-dimethyl-4-(2-methylpropyl)-oxazolidin-5-yl]methyl]-N-[(phenylmethoxy)carbonyl]-N-[2-(2-pyridinyl)ethyl]-L-leucinamide(422 mg., 0.660 mmole) in methylene chloride (3 ml.) is cooled in anice-water bath and treated with trifluoroacetic acid (6 ml.). The bathis removed and the stoppered reaction is stirred for one hour at ambienttemperature, then concentrated in vacuo and treated with tetrahydrofuran(8 ml.) and 1N hydrochloric acid (3 ml.). After 4 hours, the reaction isbasified with 15 ml. of saturated aqueous sodium bicarbonate andextracted with two 30 ml. portions of chloroform. The combined organicextracts are rinsed with brine, dried (MgSO₄) and concentrated in vacuoto yield 329 mg. of free amine.

Dicyclohexylcarbodiimide (134 mg., 0.671 mmole) is added to a mixture ofthe above amine (0.620 mmole), hydroxybenzotriazole hydrate (104 mg.,0.682 mmole) andN-[N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalanyl]-3-[(phenylmethoxy)methyl]-L-histidine(324 mg., 0.620 mmole) chilled in an ice-water bath under argon. Thereaction is kept in a refrigerator overnight, then diluted with 30 ml.of ethyl acetate, chilled in an ice-bath for 15 minutes, then filteredand diluted with more ethyl acetate. The organic extract is rinsed with10 ml. each of water, saturated aqueous sodium bicarbonate and brine,dried (MgSO₄), and concentrated in vacuo to 730 mg. of crude product.Flash chromatography on 45 g. of LPS-1 silica gel eluting with 2 columnvolumes of 2:1 chloroform:ethyl acetate followed by 20:1chloroform:methanol yields 583 mg. of N²-[3-[[N-[N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalanyl]-3-[(phenylmethoxy)methyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-N-[(phenylmethoxy)carbonyl]-N-[2-(2-pyridinyl)ethyl]-L-leucinamide; [α]_(D) =-31.9° (c=1, chloroform). TLC (silica gel, 10:1chloroform:methanol) R_(f) =0.38.

Anal. calc'd. for C₅₆ H₇₄ N₈ O₉ : C, 67.04; H, 7.44; N, 11.17 Found: C,66.48; H, 7.55; N, 11.29.

(m) N²-[3-[[N-[N-[(1,1-Dimethylethoxy)carbonyl]-L-phenylalanyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-N-[2-(2-pyridinyl)ethyl]-L-leucinamide,acetic acid salt (1:2)

To a solution of the product from part (1) (558 mg., 0.556 mmole) inmethanol (25 ml.) is added 5 ml. of water, 1.78 ml. of 1N hydrochloricacid, and 90 mg. of 20% palladium hydroxide on carbon catalyst. Themixture is hydrogenated at 1 atmosphere of hydrogen using a balloon for16 hours, then retreated with 90 mg. of fresh catalyst for 6 more hoursto completion. The reaction mixture is filtered, concentrated in vacuo,and the product is partitioned between 60 ml. of chloroform and 20 ml.of saturated sodium bicarbonate. Some ether is added to separate thelayers and the organic extract is rinsed with brine, dried (MgSO₄), andconcentrated in vacuo to afford crude product. Flash chromatography on45 g. of LPS-1 silica gel eluting with 90:20:2.5:1chloroform:methanol:water:acetic acid gives 342 mg. of N²-[3-[[N-[N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalanyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-N-[2-(2-pyridinyl)ethyl]-L-leucinamide, acetic acid salt (1:2); m.p.70°-82°; [α]_(D) =-16.6° (c=0.5, methanol). TLC (silica gel, 90:20:2.5:1chloroform:methanol:water:acetic acid) R_(f) =0.29.

Anal. calc'd. for: C₄₀ H₆₀ N₈ O₆.2C₂ H₄ O₂.1.6H₂ O: C, 58.85; H, 7.99;N, 12.48, Found: C, 58.81; H, 7.85; N, 12.50.

EXAMPLE 2(2R,3S)-[3-[[N-[(1,1-Dimethylethoxy)carbonyl]-L-phenylalanyl-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-L-leucinamide,diacetate salt (a) (2R,3S)- and(2S,3S)-N-[3-[[(1,1-Dimethylethoxy)carbonyl]amino]-5-methyl-2-hydroxyhexyl]-N-[(phenylmethoxy)carbonyl]-L-leucine,2-(trimethylsilyl)ethyl ester

The mixture of isomers obtained in Example 1(i) is separated on a WatersPrep 500 LC using two columns eluted with 25:1 petroleum ether:acetone(250 ml./min., 200 ml. fractions). Homogeneous fractions of the firstisomer eluted are pooled to give 1.53 g. of(2S,3S)-N-[3-[[(1,1-dimethylethoxy)carbonyl]amino]-5-methyl-2-hydroxyhexyl]-N-[(phenylmethoxy)carbonyl]-L-leucine,2-(trimethylsilyl)ethyl ester; [α]_(D) =-57.4° (c=1, chloroform). TLC(silica gel, 10:1 petroleum ether:acetone) R_(f) =0.24.

Anal. calc'd. for C₃₁ H₅₄ N₂ O₇ Si: C, 62.59; H, 9.15; N, 4.71, Found:C, 62.57; H, 8.87; N, 4.99.

After collecting 132 mg. of a mixture fraction, 1.72 g. of pure(2R,3S)-N-[3-[[(1,1-dimethylethoxy)carbonyl]amino]-5-methyl-2-hydroxyhexyl]-N-[(phenylmethoxy)carbonyl]-L-leucine,2-(trimethylsilyl)ethyl ester is obtained as an oil; [α]_(D) =-25.2°(c=1, chloroform). TLC (silica gel, 10:1 petroleum ether:acetone) R_(f)=0.21.

Anal. calc'd. for C₃₁ H₅₄ N₂ O₇ Si: C, 62.59; H, 9.15; N, 4.71, Found:C, 62.29; H, 9.28; N, 4.62.

(b)(2R,3S)-N-[[3-[(1,1-Dimethylethoxy)carbonyl]-2,2-dimethyl-4-(2-methylpropyl)oxazolidin-5-yl]methyl]-N-[(phenylmethoxy)carbonyl]-L-leucine,2-(trimethylsilyl)ethyl ester

The (2R,3S) isomer product from part (a) (2.42 g., 4.1 mmole) isdissolved in 72 ml. of dry methylene chloride and treated with2-methoxypropene (5.9 g., 82 mmole), followed bypyridinium-p-toluenesulfonic acid (0.206 g., 0.802 mmole). The reactionis stirred under nitrogen at room temperature for one hour and thendiluted with ether (500 ml.) and rinsed with water and brine, dried(MgSO₄), and concentrated in vacuo to give 3.1 g. of crude product.Chromatography on a Waters Prep 500 LC using two columns eluted with35:1 petroleum ether:acetone (250 ml./min., 125 ml. fractions) yields2.2 g. of(2R,3S)-N-[[3-[(1,1-dimethylethoxy)carbonyl]-2,2-dimethyl-4-(2-methylpropyl)oxazolidin-5-yl]methyl]-N-[(phenylmethoxy)carbonyl]-L-leucine,2-(trimethylsilyl)ethyl ester as an oil; [α]_(D) =-4.3° (c=1,chloroform). TLC (silica gel, 15:1 petroleum ether:acetone) R_(f) =0.44.

Anal. calc'd. for C₃₄ H₅₈ N₂ O₇ Si: C, 64.32; H, 9.21; N, 4.41, Found:C, 64.39; H, 9.40; N, 4.25.

(c)(2R,3S)-N-[[3-[(1,1-Dimethylethoxy)carbonyl]-2,2-dimethyl-4S-(2-methylpropyl)oxazolidin-5yl]methyl]-N-[(phenylmethoxy)carbonyl]-L-leucinamide

A solution of the 2-(trimethylsilyl) ethyl ester product from part (b)(1.3 g., 2.05 mmole) in dimethylformamide (17 ml.) is treated with tetran-butyl ammonium fluoride trihydrate (678 mg., 2.15 mmole), and thereaction is stirred at ambient temperature for 90 minutes. The reactionis cooled to -20° under nitrogen, then treated withisobutylchloroformate (279 μl, 2.15 mmole) and kept at -15° for 15minutes. The reaction is then treated with 6N ammonia/methanol (1.1 ml.,6.60 mmole), kept at 0° for several minutes, and then allowed to warm toambient temperature. After one hour, the reaction is worked up bytreating with 150 ml. of water, extracted with 3×300 ml. of a 1:1mixture of ethyl acetate:ethyl ether, dried, and concentrated in vacuoto give 1.1 g. of crude product. Flash chromatography on 110 g. of LPS-1silica gel eluting with 5% acetone in petroleum ether separates outstarting material and eluting with 10% acetone in petroleum ether givesthe product. The product containing fractions are pooled to give 635 mg.of(2R,3S)-N-[[3-[(1,1-dimethylethoxy)carbonyl]-2,2-dimethyl-4S-(2-methylpropyl)oxazolidin-5-yl]methyl]-N-[(phenylmethoxy)carbonyl]-L-leucinamide;[α]_(D) =+4.9° (c=1, chloroform). TLC (silica gel, 1:1 petroleumether:acetone) R_(f) =0.65.

Anal. calc'd. for C₂₉ H₄₇ N₃ O₆ : C, 65.26; H, 8.88; N, 7.87, Found: C,65.10; H, 8.87; N, 7.63.

(d)(2R,3S)-[3-[[N-[(1,1-Dimethylethoxy)carbonyl]-L-phenylalanyl-N-[(phenylmethoxy)methyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-N-[(phenylmethoxy)carbonyl]-L-leucinamide

A solution of the amide product from part (c) (349 mg., 0.65 mmole) indry methylene chloride (3.6 ml.) is cooled in an ice-water bath undernitrogen and treated with trifluoroacetic acid (1.8 ml.). The stopperedreaction is stirred at room temperature for 1.5 hours, then concentratedin vacuo and treated with tetrahydrofuran (3 ml.) and 1N aqueoushydrochloric acid (1.4 ml.). After 3 hours at room temperature, thereaction is treated with 10 ml. of saturated sodium bicarbonate andextracted with two 20 ml. portions of chloroform. The combined organicextracts are rinsed with brine, dried (MgSO₄), and concentrated in vacuoto give 270 mg. of the amino alcohol.

Dicyclohexylcarbodiimide (140 mg., 0.68 mmole) is added to a mixture ofthe above amino alcohol (0.65 mmole), hydroxybenzotriazole hydrate (99mg., 0.65 mmole), andN-[N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalanyl]-3-[(phenylmethoxy)methyl]-L-histidine(340 mg., 0.65 mmole) in dimethylformamide (5 ml.) cooled in anice-water bath under nitrogen. The reaction is allowed to warm to roomtemperature overnight, then diluted with 40 ml. of ethyl acetate and 10ml. of ether and filtered. The extract is washed with 10 ml. portions of5% potassium bisulfate, water, saturated sodium bicarbonate, water, andbrine, dried (MgSO₄), and concentrated in vacuo to give 558 mg. of crudeproduct. Flash chromatography on 35 g. of LPS-1 silica gel eluting witha gradient of 2:1 chloroform-ethyl acetate to 20:1 chloroform:methanolyields 232 mg. of(2R,3S)-[3-[[N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalanyl-N-[(phenylmethoxy)methyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-N-[(phenylmethoxy)carbonyl]-L-leucinamide;

m.p. 92°-100°; [α]_(D) =-31.8° (c=1, chloroform). TLC (silica gel, 10:1chloroform:methanol) R_(f) =0.31.

(e)(2R,3S)-[3-[[N-[(1,1-Dimethylethoxy)carbonyl]-L-phenylalanyl-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-L-leucinamide,diacetate salt

A mixture of the product from part (d) (220 mg., 0.245 mmole), 20%palladium hydroxide on carbon catalyst (75 mg.), methanol (11 ml.),water (2.2 ml.) and 1N aqueous hydrochloric acid (0.54 ml.) ishydrogenated at 1 atmosphere for 20 hours. The reaction is filtered,concentrated in vacuo to 180 mg. of crude product, and flashchromatographed on 30 g. of LPS-1 silica gel eluting with 100:20:2.5:1chloroform:methanol:water:acetic acid to give 94 mg. of(2R,3S)-[3-[[N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalanyl-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-L-leucinamide,diacetate salt; m.p. 95°-99°, then 161°-164° (dec.); [α]_(D) =-12.6°(c=0.5, methanol). TLC (silica gel; 90:20:2.5:1chloroform:methanol:water:acetic acid) R_(f) =0.23.

Anal. calc'd. for C₃₃ H₅₃ N₇ O₆.2C₂ H₄ O₂.2.3H₂ O: C, 55.18; H, 8.21; N,12.18. Found: C, 54.98; H, 7.84; N, 12.22.

EXAMPLE 3N-[(3S)-3-[[N-[(1,1-Dimethylethoxy)carbonyl]-3-[(phenylmethoxy)methyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-L-leucinamide,diacetate salt (a)N-[[(3S)-3-[(1,1-Dimethylethoxy)carbonyl]-2,2-dimethyl-4-(2-methylpropyl)oxazolidin-5-yl]-methyl]-N-[(phenylmethoxy)carbonyl]-L-leucinamide

A solution ofN-[[(3S)-3-[(1,1-dimethylethoxy)carbonyl]-2,2-dimethyl-4-(2-methylpropyl)oxazolidine-5-yl]methyl]-N-[(phenylmethoxy)carbonyl]-L-leucine,2-(trimethylsilyl)ethyl ester (379 mg., 0.597 mmole), from Example 1(j),in dimethylformamide (4 ml.) is treated with tetra-n-butyl ammoniumfluoride trihydrate (414 mg., 1.31 mmole) at room temperature underargon. After 3 hours the reaction mixture is taken up in 25 ml. of ethylacetate and 25 ml. of ether. The organic extract is rinsed with three 10ml. portions of water, brine, dried (MgSO₄), and concentrated in vacuoto give 450 mg. of crude carboxylic acid.

The above carboxylic acid (0.597 mmole) is dissolved in drytetrahydrofuran (1.8 ml.) and cooled to -25° under argon.N-methylmorpholine (60.4 g., 0.597 mmole) is added followed by the slow,careful addition of isobutylchloroformate (81.5 mg., 0.597 mmole). After5 minutes 7N methanolic ammonia (0.358 ml., 2.51 mmole) is slowly addeddropwise. The external bath temperature is allowed to warm to -10° overa period of 30 minutes, then the reaction is stoppered and left in arefrigerator overnight. The reaction is next diluted with 60 ml. of 1:1ethyl acetate:ether and rinsed with two 10 ml. portions of water, 10 ml.of 5% potassium bisulfate, water and brine, dried (MgSO₄), andconcentrated in vacuo to give 500 mg. of crude product. Flashchromatography on 30 g. of LPS-1 silica gel eluting initially with 3column volumes of chloroform, followed by 30:1 chloroform:methanolyields 313 mg. ofN-[[(3S)-3-[(1,1-dimethylethoxy)carbonyl]-2,2-dimethyl-4-(2-methylpropyl)oxazolidin-5-yl]methyl]-N-[(phenylmethoxy)carbonyl]-L-leucinamide.TLC (silica gel, 20:1 chloroform:methanol) R_(f) =0.48, 0.54.

(b)N-[(3S)-3-[[N-[(1,1-Dimethylethoxy)carbonyl]-3-[phenylmethoxy)methyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-N²-[(phenylmethoxy)carbonyl]-L-leucinamide

The amide product from part (a) (310 mg., 0.581 mmole) is dissolved in 6ml. of dry methylene chloride cooled in an ice-water bath under argonand 3 ml. of trifluoroacetic acid is added. After one hour at ice-waterbath temperature, the reaction is concentrated in vacuo and treated with6 ml. of tetrahydrofuran and 3 ml. of 1N aqueous hydrochloric acid.After 5 hours at room temperature, the reaction is treated with 15 ml.of saturated sodium bicarbonate and extracted with two 20 ml. portionsof chloroform. The organic extract is rinsed with brine, dried (MgSO₄),and concentrated in vacuo to give 253 mg. of aminoalcohol.

Dicyclohexylcarbodiimide (126 mg., 0.610 mmole) is added to a mixture ofthe above aminoalcohol (0.581 mmole), hydroxybenzotriazole hydrate (97.8mg., 0.639 mmole), andN-[(1,1-dimethylethoxy)carbonyl]-3-[(phenylmethoxy)methyl]-L-histidine(218 mg., 0.581 mmole) [prepared according to the procedure of Brown etal., J. Chem. Soc. Perkins Trans., Vol. 1, p. 2261 (1979)] indimethylformamide (5 ml.) in an ice-bath under argon. The reaction isallowed to warm to room temperature overnight. It is then diluted with30 ml. of ethyl acetate, chilled in an ice-bath for 30 minutes, thenfiltered, then diluted with an additional 30 ml. of ether, and rinsedwith two 10 ml. portions of water, two 10 ml. portions of saturatedsodium bicarbonate, water, and brine, dried (MgSO₄) and concentrated invacuo to 438 mg. of crude product. Flash chromatography on 25 g. ofLPS-1 silica gel eluting with two column volumes of 3:2 chloroform:ethylacetate followed by 15:1 chloroform:methanol yields 270 mg. ofN-[(3S)-3-[[N-[(1,1-dimethylethoxy)carbonyl]-3-[(phenylmethoxy)methyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-N²-[(phenylmethoxy)carbonyl]-L-leucinamide as a 0.7 water solvate; m.p.75°-98°; [α]_(D) =-58.6° (c=1, chloroform). TLC (silica gel, 15:1chloroform:methanol) R_(f) =0.26.

(c)N-[(3S)-3-[[N-[(1,1-Dimethylethoxy)carbonyl]-3-[(phenylmethoxy)methyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-L-leucinamide,diacetate salt

A mixture of the product from part (b) (260 mg., 0.340 mmole), methanol(6 ml.), water (0.4 ml.), acetic acid (1.2 ml.), and 10% palladium oncarbon catalyst (75 mg.) is hydrogenated at 1 atmosphere for two hours.The reaction is then filtered, concentrated in vacuo, and flashchromatographed on 25 g. of LPS-1 silica gel eluting with 100:20:2.5:1chloroform:methanol:water:acetic acid to give 186 mg. ofN-[(3S)-3-[[N-[(1,1-dimethylethoxy)carbonyl]-3-[(phenylmethoxy)methyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-L-leucinamide,diacetate salt; m.p. 109°-123°; [α]_(D) =-28.6° (c=0.5, methanol), TLC(silica gel; 90:20:2.5:1 chloroform:methanol:water:acetic acid) R_(f)=0.30.

Anal. calc'd. for C₃₂ H₅₂ N₆ O₆.2C₂ H₄ O₂.1.2H₂ O C, 57.01; H, 8.29; N,11.08. Found: C, 57.05; H, 8.08; N, 10.91.

EXAMPLE 4N-[(3S)-3-[[N-[(1,1-Dimethylethoxy)carbonyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-L-leucinamide,acetate salt

20% Palladium hydroxide on carbon catalyst (10 ml.) is added toN-[(3S)-3-[[N-[(1,1-dimethylethoxy)carbonyl]-3-[(phenylmethoxy)methyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-L-leucinamide,diacetate (148 mg., 0.195 mmole) dissolved in a mixture of methanol (10ml.), water (2.1 ml.), and 1N aqueous hydrochloric acid (0.429 ml.). Themixture is kept under 1 atmosphere of hydrogen for 18 hours. Two flashchromatographies on 20-25 g. LPS-1 silica gel eluting with 90:20:2.5:1chloroform:methanol:water:acetic acid remove byproduct. Productfractions are pooled and triturated with ether to give 83 mg. of acetatesalt. This product is dissolved in aqueous methanol and filtered througha polycarbonate millipore filtration device. Removal of solvents invacuo yields 75 mg. of triturated salt. This material is partitionedbetween chloroform and saturated aqueous sodium bicarbonate. Afterdrying (Na₂ SO₄ and MgSO₄), the chloroform extract is treated withacetic acid (0.2 ml.) and concentrated in vacuo to yield 60 mg. ofN-[(3S)-3-[[N-[(1,1-dimethylethoxy)carbonyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-L-leucinamide,acetate salt; m.p. 66°-74°; [α]_(D) =-36.4° (c=0.5, methanol). TLC(silica gel, 90:20:2.5:1 chloroform:methanol:water:acetic acid) R_(f)=0.16.

Anal. calc'd for C₂₄ H₄₄ N₆ O₅.1.6C₂ H₄ O₂.0.8H₂ O: C, 53.80; H, 8.63;N, 13.84. Found: C, 53.84; H, 8.49; N, 13.84.

EXAMPLE 5(2S,3S)-[3-[[N-[(1,1-Dimethylethoxy)carbonyl]-L-phenylalanyl-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-L-leucinamide,diacetate salt (a)(2S,3S)-N-[[3-[(1,1-Dimethylethoxy)carbonyl]-2,2-dimethyl-4-(2-methylpropyl)oxazolidin-5-yl]methyl]-N-[(phenylmethoxy)carbonyl]-L-leucine,2-(trimethylsilyl)ethyl ester

2-Methoxypropene (3.49 g., 48.4 mmole) is added to a solution of(2S,3S)-N-[3-[[(1,1-dimethylethoxy)carbonyl]amino]-5-methyl-2-hydroxyhexyl]-N-[(phenylmethoxy)carbonyl]-L-leucine,2-(trimethylsilyl)ethyl ester (1.44 g., 2.42 mmole) [prepared in Example2(a)] in dry methylene chloride (42 ml.) followed by the addition ofpyridine-p-toluenesulfonic acid (0.12 g., 0.48 mmole). The reactionmixture is stirred at ambient temperature under nitrogen for 45 minutes,the diluted with ether (350 ml.), rinsed with water and brine, dried(MgSO₄), and concentrated in vacuo to give 2.0 g. of crude product.Flash chromatography on 113 g. of LPS-1 silica gel eluting with agradient of from 100:1 to 25:1 petroleum ether:acetone yields 1.46 g. of(2S,3S)-N-[[3-[(1,1-dimethylethoxy)carbonyl]-2,2-dimethyl-4-(2-methylpropyl)oxazolidin-5-yl]methyl]-N-[(phenylmethoxy)carbonyl]-L-leucine,2-(trimethylsilyl(ethyl ester as an oil; [α]_(D) =-19.6° (c=1,chloroform). TLC (silica gel, 10:1 petroleum ether:acetone) R_(f) =0.53.

(b)(2S,3S)-N-[[3-[(1,1-Dimethylethoxy)carbonyl]-2,2-dimethyl-4-(2-methylpropyl)oxazolidin-5-yl]methyl]-N-[(phenylmethoxy)carbonyl]-L-leucinamide

A solution of the 2-(trimethylsilyl)ethyl ester product from part (a)(1.5 g., 2,36 mmole) in dimethylformamide (12 ml.) is treated withtetra-n-butyl ammonium fluoride trihydrate (1.64 g., 5.2 mmole) and thereaction is stirred at ambient temperature for 4 hours. The reaction isdiluted with 75 ml. of ether and ethyl acetate, then rinsed with 40 ml.of water (3 times) and brine, dried (MgSO₄), and concentrated in vacuoto give 1.16 g. of crude free acid.

This free acid (2.17 mmole) is dissolved in tetrahydrofuran (6.5 ml.),cooled to -25° under argon, and then treated with N-methyl morpholine(219 mg., 2.17 mmole), followed by the slow careful addition ofisobutylchloroformate (296 mg., 2.17 mmole). After five minutes, thereaction is treated carefully with 7N methanolic ammonia (1.3 ml., 9.11mmol.), warmed to -10° over a 30 minute period, then stoppered well, andstored in the refrigerator overnight. Afterward, the reaction is dilutedwith 70 ml. of ethyl acetate and ether, rinsed with water (3×20 ml.), 5%potassium bisulfate (30 ml.), water, and brine, dried (MgSO₄), andconcentrated in vacuo to give 1.12 g. of crude product. The compound ispurified by flash chromatography on 60 g. LPS-1 silica gel eluting with2 column volumes of 2:1 chloroform:ethyl acetate followed by 20:1chloroform:methanol. The product containing fractions are pooled to give1.14 g. of(2S,3S)-N-[[3-[(1,1-dimethylethoxy)carbonyl]-2,2-dimethyl-4-(2-methylpropyl)oxazolidin-5-yl]methyl]-N-[(phenylmethoxy)carbonyl]-L-leucinamide;[α]_(D) =-70.3° (c=1, methylene chloride). TLC (silica gel, 30:1chloroform:methanol) R_(f) =0.40.

Anal. calc'd for C₂₉ H₄₇ N₃ O₆.0.5H₂ O: C, 64.18; H, 8.92; N, 7.74.Found: C, 64.13; H, 8.59; N, 7.71.

(c)(2S,3S)-[3-[[N-[(1,1-Dimethylethoxy)carbonyl]-L-phenylalanyl-3-[(phenylmethoxy)methyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-N²-[(phenylmethoxy)carbonyl]-L-leucinamide

A solution of the amide product from part (b) (445 mg., 0.83 mmole) indry methylene chloride (5 ml.) is cooled in an ice-bath under nitrogenand treated with 5 ml. of distilled trifluoroacetic acid. The reactionis stoppered and stirred at room temperature. After one hour, thereaction is concentrated in vacuo, and the residue is dissolved in 4 ml.of distilled tetrahydrofuran, treated with 1N aqueous hydrochloric acid(1.8 ml., 1.8 mmole), stoppered, and stirred at ambient temperature.After 2 hours, the reaction is worked up by diluting with 10 ml. ofsaturated aqueous sodium bicarbonate, then extracted with chloroform(2×20 ml.). The organic extracts are combined, rinsed with brine, dried(MgSO₄), and concentrated in vacuo to 370 mg. of crude amino alcohol.

This amino alcohol (370 mg., 0.83 mmole) is dissolved in drydimethylformamide (6.3 ml.) then treated withN-[N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalanyl]-3-[(phenylmethoxy)methyl]-L-histidine(434 mg., 0.83 mmole) [prepared as set forth in Example 1(c)] andhydroxybenzotriazole trihydrate (127 mg., 0.83 mmole). The resultingsolution is cooled in an ice-bath under nitrogen, then treated withdicyclohexylcarbodiimide (179 mg., 0.87 mmole), stoppered andrefrigerated overnight. Afterwards, the reaction is diluted with 40 ml.of ethyl acetate and 10 ml. of ether, filtered to remove dicyclohexylurea, and the filtrate is washed with 10 ml. portions of 5% aqueouspotassium bisulfate, water, saturated aqueous sodium bicarbonate, water,and brine, dried (MgSO₄), and concentrated in vacuo to give 652 mg. ofcrude product. The compound is purified by flash chromatography on 60 g.of LPS-1 silica gel eluting with firstly 250 ml. of 2:1 chloroform:ethylacetate, secondly with 100 ml. of a 1:1 mixture of 2:1 chloroform:ethylacetate and 25:1 chloroform:methanol, and lastly with 25:1chloroform:methanol. The product containing fractions are pooled to give380 mg. of(2S,3S)-[3-[[N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalanyl-3-[(phenylmethoxy)methyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-N²-[(phenylmethoxy)carbonyl]-L-leucinamide; m.p. 90°-98°; [α]_(D) =-49.9°(c=1, chloroform). TLC (silica gel, 15:1 chloroform:methanol) R_(f)=0.25.

Anal. Calc'd. for C₄₉ H₆₇ N₇ O₉ 1H_(20:) C, 64.24; H, 7.59; N, 10.70.Found: C, 64.13; H, 7.46; N, 10.28.

(d)(2S,3S)-[3-[[N-[(1,1-Dimethylethoxy)carbonyl]-L-phenylalanyl-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-L-leucinamide,diacetate salt

A solution of the product from part (c) (350 mg., 0.39 mmole) inmethanol (17.5 ml.) and water (3.5 ml.) is treated with 1N aqueoushydrochloric acid (0.86 ml., 0.86 mmole) and 20% palladium hydroxide oncarbon catalyst (120 mg.). The reaction is treated with a balloon ofhydrogen at room temperature overnight. Afterward, the catalyst isfiltered off, and the filtrate is concentrated in vacuo to yield 350 mg.of crude product. The compound is purified by flash chromatography on 70g. of LPS-1 silica gel eluting with 100:20:2.5:1chloroform:methanol:water:acetic acid. The product containing fractionsare pooled to yield 165 mg. of partially purified product and 74 mg. offully purified product. The 165 mg. material is rechromatographed on 33g. of LPS-1 silica gel eluting with the same solvent mixture. Theproduct containing fractions are pooled and combined with the 74 mg.from the first column to give 210 mg. of(2S,3S)-[3-[[N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalanyl-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-L-leucinamide,diacetate; m.p. 130°-131°; [α]_(D) =-20.4° (c=0.5, methanol). TLC(silica gel, 90:20:2.5:1 chloroform:methanol:water:acetic acid) R_(f)=0.19.

Anal. calc'd. for C₃₃ H₅₃ N₇ O₆.2C₂ H₄ O₂.2H₂ O: C, 55.55; H, 8.19; N,12.26. Found: C, 55.55; H, 7.83; N, 12.64.

EXAMPLES 6-23

Following the procedures of Examples 1 to 5, the aminoalcohol shownbelow in Col. I is reacted with the carboxylic acid shown in Col. II togive the product shown in Col. III.

      Col. I      ##STR39##       Col. II      ##STR40##       Col. III      ##STR41##      Example R.sub.1 R.sub.2 R.sub.3 R.sub.4 R.sub.6      6     ##STR42##      CH.sub.2 CH(CH.sub.3).sub.2 CH.sub.2 CH(CH.sub.3).sub.2 NH.sub.2      ##STR43##      7     ##STR44##      CH.sub.2 CH(CH.sub.3).sub.2 CH.sub.2 CH(CH.sub.3).sub.2 NH.sub.2      ##STR45##      8     ##STR46##      CH.sub.2      CH(CH.sub.3).sub.2 CH(CH.sub.3).sub.2 NH.sub.2     ##STR47##      9     ##STR48##      CH.sub.3 CH.sub.3 NH.sub.2      ##STR49##      10     ##STR50##      ##STR51##      CH(CH.sub.3).sub.2 NH.sub.2      ##STR52##      11     ##STR53##      ##STR54##      CH.sub.2 CH(CH.sub.3).sub.2 NH.sub.2 H.sub.5 C.sub.2      12     ##STR55##      H      ##STR56##      NH.sub.2      ##STR57##      13     ##STR58##      ##STR59##      H NH.sub.2      ##STR60##      14     ##STR61##      CH.sub.2      CCl.sub.3     ##STR62##      NH.sub.2      ##STR63##      15     ##STR64##      ##STR65##      C.sub.2      H.sub.5 NH.sub.2     ##STR66##      16     ##STR67##      ##STR68##      CH(CH.sub.3).sub.2 NH.sub.2      ##STR69##      17     ##STR70##      CH.sub.2 CH(CH.sub.3).sub.2 CH.sub.2      CH(CH.sub.3).sub.2     ##STR71##      ##STR72##      18     ##STR73##      CH.sub.2      CH(CH.sub.3).sub.2     ##STR74##      NHCH.sub.3      ##STR75##      19     ##STR76##      CH.sub.2SCH.sub.3 CH.sub.2      CH(CH.sub.3).sub.2 OCH.sub.3     ##STR77##      20     ##STR78##      CH.sub.2 CH(CH.sub.3).sub.2 CH.sub.2      CH(CH.sub.3).sub.2     ##STR79##      ##STR80##      21     ##STR81##      CH.sub.2 CH(CH.sub.3).sub.2 CH.sub.2 CH(CH.sub.3).sub.2 NH.sub.2      ##STR82##      22 CH.sub.3     ##STR83##      CH.sub.3 NH.sub.2      ##STR84##      23     ##STR85##      CH.sub.2 CH(CH.sub.3).sub.2 CH.sub.2 CH(CH.sub.3).sub.2 NH.sub.2      ##STR86##

The R₁ protecting group shown in Examples 8 to 21 and 23, the R₂protecting groups shown in Examples 16 and 22, the R₃ protecting groupshown in Example 18 are removed as the last step in the synthesis as canthe R₁₁ group shown in Examples 16, 20 and 22 and the R₆ protectinggroup shown in Examples 12, 18, and 19.

In a similar manner, by employing the corresponding aminoketone of theformula ##STR87## within the procedure of Examples 6 to 23 othercompounds within the scope of the invention are obtained.

EXAMPLE 24

1000 tablets each containing the following ingredients

    ______________________________________                                        N.sup.2 --[3-[[N--[N--[(1,1-Dimethylthoxy)                                                           250 mg.                                                carbonyl]-L-phenylalanyl] -L-                                                 histidyl]amino]-2-hydroxy-5-methyl-                                           hexyl]-N--[2-(2-pyridyl)ethyl]-                                               L-leucinamide, acetic acid salt (1:2)                                         Cornstarch             100 mg.                                                Gelatin                 20 mg.                                                Avicel (microcrystalline cellulose)                                                                   50 mg.                                                Magnesium stearate      5 mg.                                                                        425 mg.                                                ______________________________________                                    

are prepared from sufficient bulk quantities by mixing N²-[3-[[N-[N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalanyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-N-[2-(2-pyridyl)ethyl]-L-leucinamide,acetic acid salt (1:2) and cornstarch with an aqueous solution of thegelatin. The mixture is dried and ground to a fine powder. The Aviceland then the magnesium stearate are admixed with granulation. Thismixture is then compressed in a tablet press to form 1000 tablets eachcontaining 250 mg. of active ingredient.

In a similar manner, tablets containing 250 mg. of the product of any ofExamples 2 to 23 can be prepared.

A similar procedure can be employed to form tablets containing 500 mg.of active ingredient.

EXAMPLE 25

Two piece #1 gelatin capsules are filled with a mixture of the followingingredients:

    ______________________________________                                        (2R,3S)--[3-[[N--[(1,1-Dimethylethoxy)                                                               500 mg.                                                carbonyl]-L-phenylalanyl-L-histidyl]                                          amino]-2-hydroxy-5-methylhexyl] -L-                                           leucinamide, diacetate salt                                                   Magnesium stearate      7 mg.                                                 Lactose                193 mg.                                                                       700 mg.                                                ______________________________________                                    

In a similar manner capsules containing 500 mg. of the product of any ofExamples 1 and 3 to 23 can be prepared.

EXAMPLE 26

An injectable solution is prepared as follows:

    ______________________________________                                        N--[(3S--3-[[N--[(1,1-Dimethylethoxy)                                                                   1000   g.                                           carbonyl]-3-[(phenylmethoxy)methyl]-                                          L-histidyl] amino]-2-hydroxy-5-                                               methylhexyl]-L-leucinamide, diacetate                                         salt                                                                          Methyl paraben            5      g.                                           Propyl paraben            1      g.                                           Sodium chloride           5      g.                                           ______________________________________                                    

The active substance, preservatives, and sodium chloride are dissolvedin 3 liters of water for injection and then the volume is brought up to5 liters. The solution is filtered through a sterile filter andaseptically filled into presterilized vials which are closed withpresterilized rubber closures. Each vial contains 5 ml. of solution in aconcentration of 200 mg. of active ingredient per ml. of solution forinjection.

In a similar manner, an injectable solution containing 200 mg. of activeingredient per ml. of solution can be prepared for the product of any ofExamples 1, 2 and 4 to 23.

EXAMPLE 27

1000 tablets each containing the following ingredients:

    ______________________________________                                        (2S,3S)--[3-[[N--[(1,1-Dimethylethoxy)                                                               500 mg.                                                carbonyl]-L-phenylalanyl-L-histidyl]                                          amino]-2-hydroxy-5-methylhexyl]-L-                                            leucinamide, diacetate salt                                                   Avicel                 300 mg.                                                Hydrochlorothiazide    14.5 mg.                                               Lactose                113 mg.                                                Cornstarch             15.5 mg.                                               Stearic Acid            7 mg.                                                                        950 mg.                                                ______________________________________                                    

are prepared from sufficient bulk quantities by slugging the(2S,3S)-[3-[[N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalanyl-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-L-leucinamide,diacetate salt, Avicel, and a portion of the stearic acid. The slugs areground and passed through a #2 screen, then mixed with thehydrochlorothiazide, lactose, cornstarch, and remainder of the stearicacid. The mixture is compressed into 950 mg. capsule shaped tablets in atablet press.

In a similar manner, tablets can be prepared containing 500 mg. of theproduct of any of Examples 1 to 4 and 6 to 23.

What is claimed is:
 1. A compound of the formula ##STR88## including apharmaceutically acceptable salt thereof wherein: A is ##STR89## R₄ is--NR-R₅ ; R₅ is hydrogen, lower alkyl, --(CH₂)_(m) -aryl or --(CH₂)_(m)-heterocyclo;m is zero, one, two, three or four; R₈ is hydrogen,##STR90## R₁₀ is hydrogen, ##STR91## R₂, R₃, R₇ and R₉ are independentlyselected from the group consisting of hydrogen, lower alkyl, halosubstituted lower alkyl, --(CH₂)_(n) --aryl, --(CH₂)_(n) --heterocyclo,--(CH₂)_(n) --OH, --(CH₂)_(n) --NH₂, --(CH₂)_(n) --SH, --(CH₂)_(n)--S-lower alkyl, ##STR92## n is an integer from 1 to 4; R₁₁ is ##STR93##R₁ hydrogen, lower alkyl, --(CH₂)_(n) --aryl, --(CH₂)_(n) --cycloalkyl,##STR94## the term lower alkyl refers to straight or branched chainradicals having up to seven carbon atoms; the term cycloalkyl refers tosaturated rings of 4 to 7 carbon atoms; the term halogen refers to Cl,Br, and F; the term halo substituted lower alkyl refers to such loweralkyl groups in which one or more hydrogens have been replaced by Cl,Br, or F groups; the term aryl refers to phenyl, 1-naphthyl, 2-naphthyl,mono substituted phenyl, 1-naphthyl, or 2-naphthyl wherein saidsubstituent is lower alkyl of 1 to 4 carbons, lower alkylthio of 1 to 4carbons, lower alkoxy of 1 to 4 carbons, halogen, hydroxy, amino,--NH-alkyl wherein alkyl is of 1 to 4 carbons, or --N(alkyl)₂ whereinalkyl is of 1 to 4 carbons, di or tri substituted phenyl, 1-naphthyl or2-naphthyl wherein said substituents are methyl, methoxy, methylthio,halogen, or hydroxy; and the term heterocyclo refers to 2-thienyl,3-thienyl, 2-furyl, 3-furyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,imidazolyl, and indolyl.
 2. A compound of claim 1 wherein:A is ##STR95##3. A compound of claim 1 wherein:A is ##STR96##
 4. A compound of claim 3wherein:R₁ is hydrogen, ##STR97## R₂ is straight or branched chain loweralkyl of 1 to 4 carbons, benzyl, phenethyl or ##STR98## R₃ is hydrogen,straight or branched chain lower alkyl of 1 to 4 carbons, benzyl orphenethyl; R₄ is --NH₂, --NH--(CH₂)_(n) --aryl or --NH--(CH₂)_(n)-heterocyclo wherein n is one, two or three, aryl is phenyl, andheterocyclo is 2-, 3- or 4-pyridyl; R₇ is ##STR99## R₈ is ##STR100## R₉is --(CH₂)_(n) -aryl wherein aryl is phenyl or 1-naphthyl and n is oneor two; and R₁₀ is ##STR101##
 5. A compound of claim 4 whereinR₁ ishydrogen; R₂ is --CH₂ --CH(CH₃)₂ ; R₃ is --CH₂ --CH(CH₃)₂ or --CH(CH₃)₂; R₄ is --NH₂ or ##STR102## R₉ is ##STR103## and R₁₀ is ##STR104##
 6. Acompound of claim 5 wherein:R₃ is --CH₂ CH(CH₃)₂ ; R₄ is ##STR105## R₇is ##STR106## and R₈ is ##STR107##
 7. The compound of claim 6, N²-[3-[[N-[N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalanyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-N-[2-(2-pyridinyl)ethyl]-L-leucinamide,acetic acid salt (1:2).
 8. A compound of claim 5 wherein:R₃ is --CH₂CH(CH₃)₂ ; R₄ is --NH₂ ; R₇ is ##STR108## and R₈ is ##STR109##
 9. Thecompound of claim 8,(2R,3S)-[3-[[N-[(1,1,-dimethylethoxy)carbonyl]-L-phenylalanyl-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-L-leucinamide,diacetate salt.
 10. The compound of claim 8,(2S,3S)-[3-[[N-[(1,1-dimethylethoxy)carbonyl]-L-phenylalanyl-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-L-leucinamide,diacetate salt.
 11. A compound of claim 5 wherein:R₃ is --CH₂ CH(CH₃)₂ ;R₄ is --NH₂ ; R₇ is ##STR110## and R₈ is ##STR111##
 12. The compound ofclaim 11,N-[(3S)-3-[[N-[(1,1-dimethylethoxy)carbonyl]-3-[(phenylmethoxy)methyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-L-leucinamide,diacetate salt.
 13. A compound of claim 5 wherein:R₃ is --CH₂ CH(CH₃)₂ ;R₄ is --NH₂ ; R₈ is ##STR112## and R₇ is ##STR113##
 14. The compound ofclaim 13,N-[(3S)-3-[[N-[(1,1-dimethylethoxy)carbonyl]-L-histidyl]amino]-2-hydroxy-5-methylhexyl]-L-leucinamide,acetate salt (1:1:6).